1. Field of the Invention
This invention relates to the stabilization of enzymes. In particular, the invention relates to stabilized enzyme compositions containing creatine kinase isoenzymes for use in diagnostic assays.
2. Description of Related Art
Following ischemic damage to cardiac tissue, a large quantity of enzymes are released into the blood stream. The detection of the presence or increased quantity of one or more of these enzymes is routinely used to confirm that an acute myocardial infarction has occurred. Three principle enzymes are used for this purpose: creatine kinase (CK), also referred to as creatine phosphokinase, serum glutamic oxaloacetic transaminase (SGOT), and lactate dehydrogenase (LDH). The rates of release of these enzymes differ, and the temporal pattern of release is of diagnostic importance. The CK level in serum begins to rise within four to six hours after the onset of chest pain. A peak CK level is seen between 12 and 24 hours, and then the level rapidly returns to the normal range within 72 to 96 hours. In comparison, peak serum levels of SGOT occur at 24 to 48 hours, and peak LDH levels occur after two to four days. Thus, the detection of increased amounts of the CK enzyme in the blood provides an early indication of myocardial infarction.
Creatine kinase is a dimer, composed of the M and B subunits which give rise to three predominant creatine kinase isoenzymes present in human tissues. The CKBB isoenzyme (or CK.sub.1) is present predominantly in brain and intestinal tissue. The CKMB isoenzyme (or CK.sub.2) is present predominantly in heart muscle and in the diaphragm and esophagus (smooth muscle). The CKMM isoenzyme (or CK.sub.3) is present in all tissues, and especially in skeletal muscle. The CKMB isoenzyme is not unique to cardiac tissue, nor is it the predominant CK isoenzyme present in cardiac tissue. As a percentage of total CK, however, the heart contains more CKMB than do other tissues. Therefore, in the absence of major muscle trauma, the appearance of CKMB in serum is used as a clinical indication of cardiac damage.
The prompt analysis of the CKMB level for the diagnosis of acute myocardial infarction has important therapeutic and economic implications. Many patients with chest pain are admitted to coronary care units and subjected to extensive and expensive evaluations. If the analysis for CKMB in serum or plasma indicates no elevation in the CKMB level for 12 to 24 hours following chest pain, the patient can be transferred from the intensive care unit.
Because differentiation between the isoenzymes of CK is clinically important, efficient and discriminatory assays for these isoenzymes are desirable. Several different methods are conventionally used for separating and measuring CK isoenzymes. Electrophoresis and column chromatography methods, which are typically used, physically separate the isoenzymes which can then be measured. However, both the electrophoretic and the column chromatographic assays are time consuming and require considerable skill.
Other analytical methods useful in the measurement of CK isoenzymes are immunoinhibition and immunoassays. These methods rely upon the interaction of CK isoenzymes and CK isoenzyme-specific antibodies. Such methods can be automated or configured to include disposable devices, thereby decreasing user time and the need for training, and increasing the speed and facility with which the assays are performed.
Typically, enzyme preparations containing known amounts of an enzyme are provided in diagnostic kits as reference samples to assure the accuracy and consistency of the assay results. For example, with an automated CKMB immunoassay, CKMB isoenzyme preparations of varying concentration can be provided for use as instrument calibrators and/or controls. To provide precision and uniformity among separate assays when conducted over a period of time, the enzyme preparations must be stable. However, because enzymes such as the CK isoenzymes are heat labile, they must be stabilized.
Conventional techniques for stabilizing an enzyme preparation include formulating the enzyme into a solid matrix by freeze-drying or lyophilizing the reagent to form a reconstitutable powder or tablet. These techniques have disadvantages. In freeze-drying, the water is removed, thereby relinquishing part of the quality control cycle to the user's dilution and reconstitution of the reagent. Other disadvantages of freeze-dried enzyme reagents include: variable irreversible inactivation during freeze-drying, and a time and/or temperature-dependent reactivation phase. In addition, freeze-dried enzyme preparations may have a relatively short stability, once reconstituted. Currently available commercial enzyme immunoassay kits containing the CKMB isoenzyme typically provide freeze-dried calibrator and control reference reagents which are stable at 2.degree.-8.degree. C. for 7-14 days after reconstitution.
Other attempts at stabilizing enzymes involve binding the enzymes to organic carrier materials such as cellulose particles, or binding the enzyme to an inorganic carrier material with reactive silanol groups as described by Messing, U.S. Pat. No. 3,556,945. Alternatively, Modrovich et al. (U.S. Pat. No. 4,652,524) describe the covalent binding of enzymes (e.g., creatine kinase) to pendant groups on carrier polymers, such as polyacrylic acid and polymethacrylic acid, to stabilize the enzyme. These methods require complex production steps, and the binding of the enzyme to the carrier can inhibit the activity of the enzyme. The addition of adenosine diphosphate to stabilize CKMM isoenzyme is disclosed in Whitner, et al., Clinical Chemistry, 28[1], 41-44, 1982.